klenchin at REMOVE_TO_REPLY.facstaff.wisc.edu
Mon May 7 12:49:04 EST 2001
"Dr. Artem Evdokimov" <eudokima at mail.ncifcrf.gov> wrote:
:There are proteins which, even with a His tag, elute at 40-60 mM
In my experience, easily 50% of all 6-his tagged proteins
elute at ~ 50 mM imidazole (pH varies 7.0-7.50, depending
:Ideally, you should try to use 'real' chromatography (i.e.
:FPLC) to monitor the progress of your elution. For weakly binding
:proteins, you would lyse the cells in say, 50 mM phosphate/300 mM NaCl
:with protease inhibitors, load the stuff on the column and elute with
:0-300 mM gradient imidazole. There should be two peaks, one at 0-30 mM
:containing all sorts of crud and one higher than that, with (mostly)
:your target protein in it.
I tried this a while ago and compared it to step elution.
Purity-wise, step was much better _if_ it includes
ridiculously thorough isocratic wash/elution step at
~ 10 mM imidazole (like, 200 ml wash of 2-5 ml
:Importantly, TRIS is NOT recommended for IMAC (in fact most amines would
:interfere, including commonly used ammonium sulphate)
Not for a single run. Once for me, it was really very convenient
to do IMAC right after AS pption. So I did a trial run to see how
His-tagged protein binds in 250 mM ammonium sulphate. It did
bind perfectly well and eluted where it usually elutes. If I am
not mistaken, Tris should be less reactive than AS. Sure, repeated
use of a column with amines will shorten its life, but for a
single run it's fine. Same for 5 mM DTT at +4C. Even chelators
like EDTA and EGTA at ~ 2 mM do not interfere appreciably with
binding (apparently, koff for Ni2+-NTA is small enough).
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